PROJECT DESCRIPTION Synovial cell sarcoma (ScS) and Ewing's sarcoma (ES) are aggressive tumors with high mortality rates in children and adolescents. These cancers are marked by well-established fusion oncogene drivers, SYT- SSX1/2 for ScS and EWSR1-FLI1 for ES, yet because the fusion proteins encode transcriptional regulators and not enzymes, these drivers are poor drug targets and these cancers are considered undruggable. The low 3-year survival rate of these patients, often under 50%, is associated with high incidences of systemic metastatic disease which respond poorly to cytotoxic drugs. The use of histone deacetylate inhibitors (HDACi) promises to increase survival based on targeting the oncogenic genes induced by the fusion oncogene products. Yet, clinical trials with HDACi have had mixed results. Identification of shared drug-sensitive driver pathways that act alone or in concert with HDACi will undoubtedly improve the survival of ScS and CCSST patients. In Aim 1, we plan to identify testable ScS and ES drug sensitivities using a pipeline that combines shRNA/CRISPRi synthetic lethality screening with bioinformatics programs that identify essentiality pathways and cognate drug susceptibilities. We will develop a seamless work pipeline that incorporates i) deconvolution analysis to match barcode (shRNA) or sgRNA clone hits with gene identification, ii) removal of general essentiality genes, iii) optimized pathway identification from dropout gene lists, iv) identification of potential drug candidates and v) in silico prioritization of drug candidates based on existing pharmacogenomic databases. We will identify which essentiality pathways match those downregulated by HDACi, with the assumption that the drugs that target HDACi-independent essentiality pathways might synergize with HDACi against ScS and ES cells (Aim 1b). We will then test the ability of essentiality pathway drugs, alone or in combination with clinically-relevant HDACi, to inhibit ScS and ES growth in vitro and in vivo (Aim 1c). In Aim 2, we will then analyze the transcriptome of clinical ScS samples to determine whether they share expression of the druggable essentiality pathways identified in Aim 1. We will then attempt to develop patient organoid cultures in order to test their sensitivity to pathway essentiality drugs from Aim 1, alone or in combination with HDACi. Taken together, data from this project will allow us in future studies to develop preclinical therapeutic studies, with the goal of increasing ScS and ES patient survival. Our success in this proposal will validate the use of this screening/bioinformatics pipeline to identify drug sensitivities in other undruggable cancers, especially those targeting pediatric populations.